The present invention relates to new polypeptides and to the genetic material permitting their expression. More especially, it relates to new polypeptides having galanin receptor activity.
Galanin is a ubiquitous neuropeptide of 29 amino acids in mammals (30 in man) which controls miscellaneous biological functions, namely (i) endocrine secretions (insulin, somatostatin, glucagon, growth hormone, etc.), (ii) muscle tone in the digestive tract, (iii) control of behaviour (food intake, perception of painful stimuli, learning, memory, pain, etc.), through a neuromodulatory effect at central nervous system level. This non-exhaustive list of the effects of galanin, most often demonstrated in animals, explains the growing interest of pharmacologists in this neuropeptide. Selective molecules (galanin agonist or antagonist) would constitute potential pharmacological agents in endocrinology, neurology and psychiatry (Bartfai et al., (1992) TIPS 13, 312-317).
A study of the mechanism of action of galanin shows that it acts via specific membrane receptors. The biochemical and molecular characterization of the receptor (Chen et al., (1993) PNAS 90, 3845-3849, Fisone et al., (1989) Eur. J. Biochem. 180, 269-276) indicates that it belongs to the family of receptors coupled to G proteins. Depending on the target tissues, the peptide inhibits adenylate cyclase, decreases intracellular calcium, blocks voltage-dependent calcium channels or activates ATP-sensitive potassium channels. Studies of structure-activity relationships, by means of C- and N-terminal fragments of galanin and of chimeric peptides, have shown that (1) irrespective of the tissue, no partial sequence of the peptide is sufficient for obtaining full activity, (2) depending on the tissues, the first two N-terminal amino acids and the C-terminal domain 16-29 of galanin are not always essential for its activity. These observations suggest the existence of subtypes of galaninergic receptors.
The present invention describes for the first time the cloning of a gene coding for a human galaninergic receptor. The present invention also describes for the first time the sequence of galaninergic receptors and their expression in recombinant cells. The present invention thus permits a better understanding of the structure of the galaninergic receptors and enables their mechanism of action to be studied more meticulously. The present invention also enables galaninergic receptors of very high purity to be obtained in large amounts, enabling functional and pharmacological studies, the production of antibodies, and the like, to be carried out. The invention also makes it possible to prepare fragments of galaninergic receptors of defined size, as well as all kinds of derivatives of galaninergic receptors. The invention also provides recombinant cells expressing galaninergic receptors or fragments of galaninergic receptors, which are usable for the screening of ligands for these receptors (agonists, antagonists, modulators, and the like). The DNA sequences of the invention also make it possible to produce probes capable of detecting any irregularity in the expression of a galaninergic receptor (non-expression, mutation, polymorphism, and the like) in biological samples. These probes can also be used for the cloning by hybridization of any other cDNA coding for a galaninergic receptor, from tissues of diverse origins, as indicated later.
Hence a first subject of the invention lies in a nucleotide sequence coding for a polypeptide having galaninergic receptor activity. For the purposes of the invention, galaninergic receptor comprises, in particular, all the potential subtypes.
More preferably, the nucleotide sequence according to the invention is chosen from:
(a) all or part of the nucleotide sequence SEQ ID No. 1 or of its complementary strand,
(b) any sequence hybridizing with a sequence (a) and coding for a polypeptide having galaninergic receptor activity, and
(c) the sequences derived from the sequences (a) and (b) as a result of the degeneracy of the genetic code.
A very special embodiment of the invention is represented by a nucleotide sequence comprising all or part of the nucleotide sequence SEQ ID No. 1 or of its complementary strand.
The different nucleotide sequences of the invention may be of artificial origin or otherwise. They can be genomic, cDNA or RNA sequences, hybrid sequences or synthetic or semi-synthetic sequences. These sequences may be obtained, for example, by the screening of DNA libraries (cDNA library, genomic DNA library) by means of probes produced on the basis of the sequence SEQ ID No. 1. Such libraries may be prepared from cells of different origins by standard techniques of molecular biology known to a person skilled in the art. The nucleotide sequences of the invention may also be prepared by chemical synthesis, in particular according to the phosphoramidite method, or alternatively by mixed methods including the chemical or enzymatic modification of sequences obtained by the screening of libraries.
The nucleotide sequences of the invention may be used for the production of galaninergic polypeptides. The term galaninergic polypeptide denotes any polypeptide having galaninergic receptor activity, and any fragment or derivative of such a polypeptide. For the production of galaninergic polypeptides, the portion coding for the said polypeptide is generally placed under the control of signals permitting its expression in a cell host. The choice of these signals (promoters, terminators, and the like) may vary in accordance with the cell host used. To this end, the nucleotide sequences of the invention may form part of a vector, which can be autonomously replicating or integrative. More especially, autonomously replicating vectors may be prepared using sequences which are autonomously replicating in the chosen host. As regards integrative vectors, these may be prepared, for example, using sequences which are homologous to certain regions of the host""s genome, permitting integration of the vector by homologous recombination. The cell hosts which can be used for the production of the galaninergic polypeptides of the invention by methods employing recombination are either eukaryotic or prokaryotic hosts. Among suitable eukaryotic hosts, animal cells, yeasts or fungi may be mentioned. In particular, as regards yeasts, yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be mentioned. As regards animal cells, COS, CHO, C127, NIH-3T3, and the like, cells may be mentioned. Among fungi, Aspergillus ssp. or Trichoderma ssp. may be mentioned more especially. As prokaryotic hosts, it is preferable to use the following bacteria: E.coli, Bacillus or Streptomyces.
The nucleotide sequences of the present invention can also be used in the pharmaceutical field, either for the production of antisense sequences which can be used in the context of a gene therapy, or for the production of probes permitting the detection, by hybridization experiments, of the expression of galaninergic receptors in biological samples and the demonstration of genetic abnormalities (polymorphism, mutations) or of aberrant expressions.
The inhibition of the expression of certain genes by antisense sequences has proved to be a promising strategy in the control of the activity of a gene. Antisense sequences are sequences whose transcription product is complementary to the coding strand of a given gene and is, as a result, capable of hybridizing specifically with the transcribed mRNA or with the gene, inhibiting its transcription or its translation into protein. Thus, a subject of the invention is the antisense sequences capable of at least partially inhibiting the production of galaninergic polypeptides as are defined above. Such sequences can consist of all or part of the nucleotide sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides of the invention. Such sequences may be obtained from the sequence SEQ ID No. 1, by fragmentation, and the like, or by chemical synthesis.
As mentioned above, the invention also makes it possible to produce nucleotide probes, synthetic or otherwise, capable of hybridizing with the nucleotide sequences defined above which code for galaninergic polypeptides of the invention, or with the corresponding mRNAs. Such probes may be used in vitro as a diagnostic tool for detecting the expression of a galaninergic receptor, or alternatively for demonstrating genetic abnormalities (incorrect splicing, polymorphism, point mutations, and the like). In view of the large number of activities of the endogenous ligands for the galaninergic receptors, the probes of the invention may thus enable neurological, cardiovascular, endocrinological or psychiatric disorders to be identified. These probes may also be used for demonstrating and isolating homologous nucleic acid sequences coding for galaninergic polypeptides as are defined above, from other cellular sources and preferably from cells of human origin. Although the present application is illustrated more especially by a clone designated GalR1, the biochemical and immunological studies described in the literature indicate the existence of subtypes of galaninergic receptors. The probes of the invention enable these different subtypes to be isolated by techniques known to a person skilled in the art (see Example 4). The probes of the invention generally contain at least 10 bases, and they can contain up to the whole of the sequence SEQ ID No. 1 or of its complementary strand. Preferably, these probes are labelled prior to their use. For this purpose, different techniques known to a person skilled in the art may be employed (radioactive labelling, enzyme labelling, and the like). The hybridization conditions under which these probes may be used are indicated in the general cloning techniques below, as well as in the examples.
A subject of the invention is also any polypeptide resulting from the expression of a nucleotide sequence as defined above. Preferably, it is a polypeptide comprising all or part of the peptide sequence SEQ ID No. 2 or of a derivative of the latter.
For the purposes of the present invention, the term derivative denotes any molecule obtained by modification of a genetic and/or chemical nature of the peptide sequence SEQ ID No. 2. Modification of a genetic and/or chemical nature may be understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for different purposes, such as, in particular, that of increasing the affinity of the peptide for its ligand(s), that of improving its levels of production, that of increasing its resistance to proteases, that of increasing and/or modifying its activity or that of endowing it with new pharmacokinetic and/or biological properties. Among the derivatives resulting from an addition, chimeric polypeptides containing an additional heterologous portion linked to one end may be mentioned as an example. The term derivative also comprises polypeptides homologous to the polypeptide SEQ ID No. 2 originating from other cellular sources, and in particular from cells of human origin, or from other organisms, and possessing an activity of the same type. Such homologous polypeptides comprise, in particular, the different subtypes of galaninergic receptors. They may be obtained, in particular, by hybridization experiments as described in the examples.
Preferably, the polypeptides of the invention are polypeptides possessing the capacity to bind galanin or a variant of galanin. Still according to a preferred embodiment, the polypeptides of the invention are capable of being recognized by antibodies that recognize the complete peptide sequence SEQ ID No. 2. Such antibodies may be generated by any technique known to a person skilled in the art, using the polypeptides described in the present application as antigens.
As indicated in the examples, these polypeptides may be expressed in different cell types to form functional galaninergic receptors.
The polypeptides of the invention may be obtained by expression in a cell host of a nucleotide sequence as described above, by chemical synthesis on the basis of the sequence SEQ ID No. 2 using techniques known to a person skilled in the art, or by a combination of these techniques.
Another subject of the invention relates to recombinant cells capable of expressing at their surface a polypeptide having galaninergic receptor activity. These cells may be obtained by introducing a nucleotide sequence as defined above, and then culturing the said cells under conditions for expression of the said sequence.
The recombinant cells according to the invention can be either eukaryotic or prokaryotic cells. Among suitable eukaryotic cells, animal cells, yeasts or fungi may be mentioned. In particular, as regards yeasts, yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be mentioned. As regards animal cells, COS, CHO, C127, NIH-3T3, and the like, cells, may be mentioned. Among fungi, Aspergillus ssp. or Trichoderma ssp. may be mentioned more especially. As prokaryotic cells, it is preferable to use the following bacteria: E.coli, Bacillus or Streptomyces. The cells thereby obtained may be used to measure the capacity of different molecules to behave as a ligand for the galaninergic receptors or as a modulator of the activity of these receptors. More especially, they may thus be used in a method for demonstrating and isolating ligands for the galaninergic receptors or modulator of the activity of these receptors, and more preferably agonists and antagonists.
Hence another subject of the invention relates to a method for demonstrating and/or isolating ligands for the galaninergic receptors, according to which the following steps are carried out:
a molecule or a mixture containing different molecules, where appropriate unidentified, is brought into contact with a recombinant cell as described above expressing at its surface a polypeptide having galaninergic receptor activity, under conditions permitting interaction between the said polypeptide and the said molecule in the case where the latter were to possess an affinity for the said polypeptide, and
the molecules bound to the said polypeptide are detected and/or isolated.
In a particular embodiment, this method of the invention is adapted to the demonstration and/or isolation of agonists and antagonists of galanin for the galaninergic receptors.
Another subject of the invention relates to a method for demonstrating and/or isolating modulators for the galaninergic receptors, according to which the following steps are carried out:
a molecule or a mixture containing different molecules, where appropriate unidentified, is brought into contact with a recombinant cell as described above expressing at its surface a polypeptide having galaninergic receptor activity, in the presence of the endogenous ligand for the said receptor, under conditions permitting interaction between the said polypeptide and its ligand, and
the molecules capable of modulating the activity of the ligand with respect to the said polypeptide are detected and/or isolated.
In a particular embodiment, this method of the invention is adapted to the demonstration and/or isolation of modulators of the activity of galanin with respect to the galaninergic receptors.
Another subject of the invention relates to the use of a ligand or a modulator identified and/or obtained according to the method described above, as a medicinal product. Such ligands or modulators can, in effect, enable certain disorders associated with the galaninergic receptors to be treated. In particular, since the galaninergic receptors are mediators of an analgesic effect, the agonists for these receptors may be used to decrease pain sensations. Other activities of the galaninergic receptors have been mentioned in the introduction.
The invention also relates to any medicinal product comprising as active principle at least one molecule which acts on a receptor of the invention. Preferably, the molecule is a ligand or a modulator identified and/or isolated according to the method described above.
Other advantages of the present invention will become apparent on reading the examples which follow, which are to be regarded as illustrative and non-limiting.